Messenger RNAs are exported from the nucleus as large ribonucleoprotein complexes (mRNPs). To date, proteins implicated in this process

Vertebrate TAP and its yeast ortholog Mex67p are involved in the export of messenger RNAs from the nucleus. TAP has also been implicated in the export of simian type D viral RNAs bearing the constitutive transport element (CTE). Although TAP directly interacts with CTE-bearing RNAs, the mode of interaction of TAP/Mex67p with cellular mRNAs is different from that with the CTE RNA and is likely to be mediated by protein-protein interactions. Here we show that Mex67p directly interacts with Yra1p, an essential yeast hnRNP-like protein. This interaction is evolutionarily conserved as Yra1p also interacts with TAP. Conditional expression in yeast cells implicates Yra1 p in the export of cellular mRNAs. Database searches revealed that Yra1p belongs to an evolutionarily conserved family of hnRNP-like proteins having more than one member in Mus musculus, Xenopus laevis, Caenorhabditis elegans, and Schizosaccharomyces pombe and at least one member in several species including plants. The murine members of the family directly interact with TAP. Because members of this protein family are characterized by the presence of one RNP-motif RNA-binding domain and exhibit RNA-binding activity, we called these proteins REF-bps for RNA and export factor binding proteins. Thus, Yra1p and members of the REF family of hnRNP-like proteins may facilitate the interaction of TAP/Mex67p with cellular mRNAs.

Vertebrate TAP (also called NXF1) and its yeast orthologue, Mex67p, have been implicated in the export of mRNAs from the nucleus. The TAP protein includes a noncanonical RNP-type RNA binding domain, four leucine-rich repeats, an NTF2-like domain that allows heterodimerization with p15 (also called NXT1), and a ubiquitin-associated domain that mediates the interaction with nucleoporins. Here we show that TAP belongs to an evolutionarily conserved family of proteins that has more than one member in higher eukaryotes. Not only the overall domain organization but also residues important for p15 and nucleoporin interaction are conserved in most family members. We characterize two of four human TAP homologues and show that one of them, NXF2, binds RNA, localizes to the nuclear envelope, and exhibits RNA export activity. NXF3, which does not bind RNA or localize to the nuclear rim, has no RNA export activity. Database searches revealed that although only one p15 (nxt) gene is present in the Drosophila melanogaster and Caenorhabditis elegans genomes, there is at least one additional p15 homologue (p15-2 [also called NXT2]) encoded by the human genome. Both human p15 homologues bind TAP, NXF2, and NXF3. Together, our results indicate that the TAP-p15 mRNA export pathway has diversified in higher eukaryotes compared to yeast, perhaps reflecting a greater substrate complexity. mRNAs are exported from the nucleus as large ribonucleoprotein complexes (mRNPs). To date, proteins directly implicated in this process include several nucleoporins and RNA binding proteins (hnRNPs), an RNA helicase of the DEADbox family (Dbp5), and the nuclear pore complex (NPC)-associated proteins Gle1p, TAP and Mex67p, and RAE1 (also called Gle2p) (reviewed in references 22, 28, and 32). Mex67p is essential for mRNA export in Saccharomyces cerevisiae, while RAE1 is essential for mRNA export in Schizosaccharomyces pombe (9,27,36). Their vertebrate homologues, TAP and RAE1, have also been implicated in the export of cellular mRNAs (6,8,12,15,20,31).We identified TAP as the cellular factor which is recruited by the constitutive transport element (CTE) of simian type D retroviruses to promote nuclear export of their genomic RNAs (12). In Xenopus oocytes, titration of TAP with an excess of CTE RNA prevents cellular mRNAs from exiting the nucleus (12,30,33). This suggests a role for this protein in the export of cellular mRNA.Considerable progress has been made in defining TAP structural and functional domains (see Fig. 1) and in identifying its binding partners. TAP partners include various nucleoporins (4, 17); p15 (also called NXT1), a protein related to nuclear transport factor 2 (NTF2) (7, 17); transportin, which mediates TAP nuclear import (4); and several mRNP-associated proteins, such as E1B-AP5, RAE1 (4), and members of the Yra1p/ REF family of proteins (37, 39). Binding of TAP to these mRNP-associated proteins is mediated by its N-terminal domain (residues 1 to 372) (4, 39). This domain includes a noncanonical RNP-type RNA binding domain (RBD)...

TAP-p15 heterodimers have been implicated in the export of mRNAs through nuclear pore complexes (NPCs). We report a structural analysis of the interaction domains of TAP and p15 in a ternary complex with a Phe-Gly (FG) repeat of an NPC component. The TAP-p15 heterodimer is structurally similar to the homodimeric transport factor NTF2, but unlike NTF2, it is incompatible with either homodimerization or Ran binding. The NTF2-like heterodimer functions as a single structural unit in recognizing an FG repeat at a hydrophobic pocket present only on TAP and not on p15. This FG binding site interacts synergistically with a second site at the C terminus of TAP to mediate mRNA transport through the pore. In general, our findings suggest that FG repeats bind with a similar conformation to different classes of transport factors.

Dbp5 is the only member of the DExH/D box family of RNA helicases that is directly implicated in the export of messenger RNAs from the nucleus of yeast and vertebrate cells. Dbp5 localizes in the cytoplasm and at the cytoplasmic face of the nuclear pore complex (NPC). In an attempt to identify proteins present in a highly enriched NPC fraction, two other helicases were detected: RNA helicase A (RHA) and UAP56. This suggested a role for these proteins in nuclear transport. Contrary to expectation, we show that the Drosophila homolog of Dbp5 is not essential for mRNA export in cultured Schneider cells. In contrast, depletion of HEL, the Drosophila homolog of UAP56, inhibits growth and results in a robust accumulation of polyadenylated RNAs within the nucleus. Consequently, incorporation of [35S]methionine into newly synthesized proteins is inhibited. This inhibition affects the expression of both heat-shock and non-heat-shock mRNAs, as well as intron-containing and intronless mRNAs. In HeLa nuclear extracts, UAP56 preferentially, but not exclusively, associates with spliced mRNAs carrying the exon junction complex (EJC). We conclude that HEL is essential for the export of bulk mRNA in Drosophila. The association of human UAP56 with spliced mRNAs suggests that this protein might provide a functional link between splicing and export.

The constitutive transport element (CTE) of the simian type D retroviruses overcomes nuclear retention and allows nuclear export of unspliced viral RNAs by recruiting TAP, a host factor which is thought to be required for export of cellular mRNAs. In this report, we show that the first 372 amino acid residues of TAP, comprising a stretch of leucine-rich repeats, are both necessary and sufficient for binding to the CTE RNA and promoting its export to the cytoplasm. Moreover, like the full-length protein, this domain migrates to the cytoplasm upon nuclear co-injection with the CTE RNA. Together, these results indicate that the CTE-binding domain includes the signals for nuclear export. We also describe a derivative of TAP that bears a triple amino acid substitution within the CTE-binding domain and substantially reduces the export of mRNAs from the nucleus. This provides further evidence for a role for TAP in this process. Thus, the CTE-binding domain of TAP defines a novel RNA-binding motif which has dual functions, both recognizing the CTE RNA and interacting with other components of the nuclear transport machinery.

No abstract

Metazoan TAP and its yeast orthologue Mex67p are members of an evolutionarily conserved protein family, the NXF family, implicated in the export of messenger RNA from the nucleus (1). Mex67p, the Saccharomyces cerevisiae NXF homologue, and the Caenorhabditis elegans protein NXF1 are essential for the export of bulk polyadenylated RNAs to the cytoplasm (2, 3), whereas human TAP (also called Hs NXF1) has been directly implicated in the export of simian type D retroviral RNAs bearing the constitutive transport element (CTE) 1 (4). In Xenopus laevis oocytes, titration of TAP with an excess of CTE RNA prevents cellular mRNAs from exiting the nucleus (4 -6), strongly suggesting a role for TAP in mRNA nuclear export, but direct evidence has so far remained elusive.Members of the NXF family of proteins have a conserved modular domain organization consisting of a non-canonical RNP-type RNA-binding domain (RBD), a leucine-rich repeat (LRR) domain, a middle region showing significant sequence similarity to nuclear transport factor 2 (the NTF2-like domain) and a C-terminal ubiquitin-associated (UBA)-like domain ( Fig. 1 and Refs. 1, 7, and 8). The LRR and the NTF2-like domains are the most conserved features of NXF proteins, whereas the RBD and the C-terminal UBA-like domain are not always present in NXF proteins (1, 3).The N-terminal half of TAP includes the LRR domain, the RBD, and a less conserved region upstream of the RBD (fragment 1-372, Fig. 1). This protein fragment exhibits general RNA binding affinity and mediates binding to several mRNA-associated proteins such as E1B-AP5 (9) and members of the Yra1p/REF protein family (10, 11). Furthermore, the RBD of TAP is required in cis to the LRR domain for specific binding to the CTE RNA (7). Hence, the RBD and the LRR domain are essential for TAP-mediated export of CTE-containing cargoes (1,7,12,13). Mutations within the LRR domains of TAP and Mex67p have been reported to affect cellular mRNA export (11, 12), but these mutations involve residues that have important structural roles and their substitution probably results in nonspecific structural aberrations.The NTF2-like domain of metazoan NXFs mediates binding to a protein known as p15 or NXT. p15 is also related to NTF2 (8, 15, 16) but unlike NTF2, which forms homodimers, p15 heterodimerizes with the NTF2-like domain of NXF proteins (1, 8). The human genome encodes at least two p15 homologues, p15-1 and p15-2, and both interact with TAP (1). The NTF2-like domain also occurs in Schizosaccharomyces pombe and S. cerevisiae Mex67p, although there is no obvious p15 homologue encoded by the yeast genome (8,15,16). In S. cerevisiae Mex67p, this domain is implicated in the interaction with a protein known as Mtr2p (14,17). Prediction of Mtr2p secondary structure and the observation that co-expression of human TAP and p15 in S. cerevisiae partially restores growth of a strain carrying the otherwise lethal mex67/mtr2 double knockout, suggest that Mtr2p may be a p15 functional analogue (8,16).A C-terminal fragment of TAP, the NP...